Fig 2: miR-138-5p regulates the heart failure process via SIRT1-regulated p53 signaling. AC-16 and HCM were induced with H2O2 for 6 h. (A) Upregulated NPPB level. (B) Mean fluorescence intensity of reactive oxygen species. (C) Apoptosis proportion. (D) Relative mRNA levels of SIRT1, p53 and NPPB. (E) Protein levels of SIRT1, p53 and acetyl-p53. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; NC, negative control; SIRT1, sirtuin 1; H2O2, hydrogen peroxide; NPPB, natriuretic peptide precursor B; NS, not significant.

Fig 3: miR-138-5p targets SIRT1. (A) The binding sites in the 3′-UTR of miR-138-5p and SIRT1 were predicted using TargetScan. (B) The direct binding between miR-138-5p and SIRT1 was analyzed by the dual-luciferase reporter assay. ***P<0.001. miR, microRNA; UTR, untranslated region; SIRT1, sirtuin 1; NS, not significant.

Fig 4: Epigenetic modulation in 1321N1 cells untreated and treated for 24 h with different concentrations of Rapha Myr® extract depending on each cell-based assay protocol. (A) Global DNA methylation evaluated by Methy-sens comet assay. (B) Changes in mRNA level of DNA methyltransferase1 (DNMT1). Western blot of mitochondrial Sirt3 (C) and Sirt5 (D). Western blot of nuclear Sirt6 (E), Sirt7 (F), Sirt1 and pSirt1 (G), and pSirt1/Sirt1 ratio (H). Each bar graph shows the relative expression of the respective protein level. (I) Nuclear sirtuins activity expressed as OD/min/mg proteins. (J) Representative immunoblotting image of each protein examined. All values are mean ± SD of three experiments in triplicate. * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control.

Fig 5: CD38 overexpression regulates Rab7 by a NAD-dependent mechanism. (A) The levels of NAD in the heart tissues of adult mice and neonatal CMs were evaluated. (B) Relative mRNA expression of Rab7 and PLEKHM1. (C,D) Representative immunoblots and quantitative results showing the expression of Rab7 and PLEKHM1. (E) Inhibition of CD38 induced Sirt1 activity in the heart tissues of mice after burn treatment and in CMs subjected to H/I. (F) FOXO1 was hyperacetylated in response to CD38 knockdown. The lower panel shows representative Western blots of whole-CM homogenate (input) or protein precipitates. The upper panel shows the densitometry results (as the fold changes over the Ctrl value) for acetylated proteins normalized to total proteins. IB, immunoblotting; IP, immunoprecipitation. (G,H) The protein expression levels of Rab7 were analyzed by immunoblotting. The data are shown as the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not statistically significant. The P-values were derived from one-way ANOVA with Bonferroni’s posttest.